HU191858B - Process for production of stabil plurilamerrical vesicula - Google Patents

Abstract

The present invention provides stable vesicles of 100 to 10,000 nm which are substantially free of multilamellar, unilamellar, and reverse phase aqueous vesicles. The vesicles of the present invention comprise a few hundred or so lipid bilayers that surround aqueous active ingredients or other compounds. The process according to the invention comprises dispersing at least one amphipathic lipid in an amount of from 20 to 3000 mg / ml, preferably a phospholipid, optionally in an organic solvent containing an antioxidant, at a reaction temperature below the boiling point of the organic solvent; The aqueous phase containing 20 to 50% by weight of the active ingredient or other compound is mixed in a ratio of 3: 1 to 40: 1, then 111, the aqueous phase is emulsified and simultaneously the organic solvent is evaporated from the biphasic system. -1-

Description

The present invention relates to the production of plurilamellar vesicles having a size of 100 to 10,000 nm which are free of multilamellar, unilamellar and reverse-phase evaporative vesicles. The vesicles produced by the process of the present invention comprise a few to more than a hundred ordered lipid bilayers of supramolecular structure surrounding the aqueous inclusions containing a vaccine.

In the process of the invention

1. dispersing at least one amphipathic lipid of 20 to 3000 mg / ml, preferably a phospholipid, in an organic solvent, optionally containing an antioxidant, at a reaction temperature below the boiling point of the organic solvent,

11. mixing the resulting dispersion with an aqueous phase containing from 20% to 50% by weight of the active compound or other compound in a ratio of 3: 1 to 40: 1, followed by

111, the aqueous phase is emulsified and the organic solvent is simultaneously evaporated from the biphasic system.

The compositions of the invention may be used in a variety of applications including delivery systems and targeted delivery systems. Examples of use of such compositions in the treatment of brucellosis, ocular infections and lymphocyte meningitis viral infections are described herein.

Liposomes are fully closed bilayer membranes containing an integrated aqueous phase. Liposomes can be variants of unilamellar vesicles (simple bilayer membrane) or multilamellar vesicles (bulbous structures consisting of concentric membrane bilayers separated by a layer of water).

Liposome preparations were first described by J. Mol. Biol. 13: 238-252 (1965), Bangham et al. The process involves suspending phospholipids in an organic solvent and removing the solvent to obtain the phospholipid in a waxy form. After the addition of an appropriate amount of aqueous phase, the reaction mixture "swells" and the liposomes formed from the multilamellar vesicles are mechanically dispersed. In the resulting bilayer membrane structure, the hydrophobic (non-polar) "tail" of the lipid is oriented toward the center of the bilayer, while the hydrophilic (polar) "head" is oriented toward the aqueous phase. The term multilamellar vesicle is hereinafter referred to as MLVs. The above procedure is the basis for the development of small ultrasound-treated unilamellar vesicles (hereinafter referred to as SUVs) described by Papahadjapoulos and Miller in Biochim. Biophys. Acta. 1967 135: 624-638. However, these "classical liposomes" have several disadvantages, so that very small volumes of water per mole of lipid can be incorporated and these liposomes are hardly suitable for encapsulation of large macromolecules.

In attempts to increase the volume of the incorporated material, inverse micelles or Eposomic precursors were first formed 2, i.e. vesicles in which the aqueous phase is surrounded by a single layer of lipid molecules such that polar head groups point towards the aqueous phase. Liposome precursors can be formed by adding the aqueous solution to be incorporated into an organic solvent in an ultrasonic mixer prepared in a polar lipid solution. The Eposomic precursors are further concentrated in the presence of half Εριά. The resulting liposome, which contains an aqueous phase incorporated into a lipid bilayer, is dispersed in an aqueous phase. This procedure is described in U.S. Patent No. 4,224,179. U.S. Pat.

No. 4,235,871. US patent discloses maximizing installation efficiency. As described herein, a "reverse-phase evaporation process" is used to produce oEgolamellar Epid vesicles known as reverse-phase evaporation vesicles (hereinafter referred to as "REVs"). In this method, the aqueous material to be incorporated is added to a mixture of a polar Epid in an organic solvent. The resulting ho25 mogen water-in-oil emulsion is further converted into a gel by evaporation of the organic solvent and then converted into a suspension by dispersing the gel-like mixture in an aqueous medium. The REVs formed are predominantly composed of unilamellar vesicles and some oEgolamellar vesicles, which are characterized by only a few concentric bilayers and a large internal water space. The REVs thus produced exhibited similar permeability properties to MLVs and SUVs (see Szoka and Papahadjopoulos, 1978, Proc. Natl. Acad. Sci. USA 75: 41944198).

Eposomes, which contain a number of compounds, can be produced in this way, but their stability remains Emital during storage. The reduction in stability results in the cleavage of the incorporated compounds from the Eposomes into the surrounding media. Reduced lubrication of the stability causes liposome contamination as other substances enter the Eposome from the surrounding medium. Conventional liposomes thus have a very short shelf life. In attempts to increase stability, certain substances, hereinafter referred to as "stabilizers", have been incorporated into the liposome membrane that affect the physical properties of the Epid bilayer (e.g. steroid groups). However, the vast majority of these materials are relatively expensive and the production of such liposomes is not economical.

Attempts have been made to solve the problems of storage of conventional liposomes with many compounds, but these have not been incorporated into these vesicles. MLVs can only be produced under the above conditions at the transition temperature of the lipid membrane phase. This precludes the incorporation of thermosensitive molecules into liposomes whose phospholipid pid moieties exhibit significant properties.

191,858 but include long and highly saturated side chains.

Applying hpo Variants!

Therapeutic applications of liposomes are described in Liposomes: From Physical Structures to Therapeutic Applications, Knight, ed. Elsevier, North-Holland Biomedical Press, 1981, new literature. The potential of such membrane vesicles to be used as therapeutic systems has been the subject of numerous publications, but many open questions remain regarding the problems of such systems. (See, for example, U.S. Patent Nos. 3,993,754 and 4,445,410.)

In a liposome therapeutic system, the active ingredient is incorporated during liposome formation and subsequently administered to the subject. The active ingredient may optionally be soluble in water or in a non-polar solvent. Typically, such inventions are disclosed in U.S. Patent No. 4,235,871. and U.S. Patent No. 4,224,179. U.S. Patents. 2

Many problems with extended release therapeutic systems providing extended drug action remain to be clarified. In these cases, the potency of the drug increases, but fewer routes of administration are possible. 2 Difficulties in the in vivo use of liposome formulations include:

1. Substances incorporated into liposomes decompose when liposomes are incubated in body fluids. This phenomenon can be attributed to liposomal phospholipid removal induced by high density lipoprotein (HDL) in plasma or other liposomal membrane degradation caused by phospholipases for other reasons.

In vivo degradation of liposomes 4 results in almost complete release of the content of the bposome in a very short period of time, thus rendering the sustained release of drug unavailable.

2. When very stable liposomes are used in vivo (that is, when liposomes are not degraded at all by incubation in body fluid), the liposome content is not dissolved to the required extent. As a result, such stable liposomes are ineffective when used as a carrier in these therapeutic systems, since the sustained release and the ability to release liposome content are not achieved at the appropriate time. Maintaining stability in body fluids until internalization of liposomes with infected cells is critical for the treatment of an intracellularly infected?

3. Economics of liposome carrier used in therapeutic systems. £

No. 4,686,183. U.S. Pat. No. 4,102,125 discloses an improved method of ketmotherapy of bposome capsules containing anti-leishmanial agents for the treatment of leishmanial infections. The bposomes used in chemotherapy contain a number of stabilizers that increase the in vivo stability of liposomes. However, as mentioned above, these stabilizers are very expensive and the preparation of liposomes containing such substances is not economical.

4. Difficulties in using bposomes as carriers in therapeutic systems render them unsuitable for curing a disease. For example, when bposomes are incorporated into target cells or phagocyte cells (e.g., reticuloendothelial cells), they are rapidly released from the system, so that the incorporated drug is much less effective against the diseased cell than the RES. After phagocytosis, the liposomal contents are stored within the bosomes of the phagocytic cells. In many cases, the cleavage enzymes within the lysosomes will cleave or deactivate the incorporated compound by altering its structure or cleaving the compound at its active sites. In addition, bposomes cannot dissolve at the proper dose due to insufficient efficacy of the active compound being incorporated into the vesicles.

The bposomes are also used as a membrane system model by researchers and used as a "target cell" to supplement the medium for immunoassays. However, a significant factor in such assays is that the liposome membrane is not degraded upon incubation in serum, since these assays measure the release of liposome contents, which signal serum complement function induced by the immune complex, including certain immunoglobulins (e.g., IgM and certain IgG molecules). .

The present invention relates to the production of a novel and significantly improved type of lipid vesicles, hereinafter referred to as stable plurylamellar vesicles (SPLVs). Not only does SPLVs differ in their structure from multilamellar vesicles (MLVs), but they are produced by a process different from that of MLVs, and SPLVs have unique properties compared to MLVs and exhibit many and varied advantages. As a result of these differences, SPLVs do not suffer from the disadvantages of known bpid vesicles.

Synthesis of SPLVs results in heterogeneous treatment of bpid vesicles. Experimental evidence supports that the lipids of SLPVs are present in a novel supramolecular structure.

Many lipid vesicles contain a large number of bilayers, in many cases the number of bilayers is slightly over 100. It is presumed that this high degree of stratification causes the surprising properties of SPLVs, although these explanations are theoretical.

SPLVs have the following properties:

1. suitable for the treatment of certain diseases which cannot be treated by other methods,

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2. very high stability when stored in buffer,

3. Resistant to aggressive physiological environments

4. ensures high efficiency of incorporated materials,

5. stays in the tissues or cells for a long time,

6. the ability to slowly inject the incorporated active substances into body fluids, ¹

7. directing liposome contents through the cytosol to the target cell,

8. production is economical,

9. active ingredients in vivo, biologically active formájá- Ban ¹ delivers.

Because of its properties above, SPLVs are particularly useful in in vivo conditions as carriers for therapeutic systems, as they are resistant to environmental influences and are capable of sustained release ⁴ . Methods for in vivo release of biologically active compounds of SPLVs, as well as treatment of diseases such as infectious medication, are described in the present disclosure.

Figure Legend

Figure 1;

It shows the difference in membrane stability of MLVs and SPLVs treated with different concentrations of urea, expressed as a percentage of degradation, graphically illustrated.

í

Figure 2:

It illustrates the retention of Epid and aqueous phases of SPLVs in murine ophthalmic tissue and prolonged release of gentamicin encapsulated in SPLVs in vivo. z

Figure 3:

Comparison of electron spin resonance absorption spectra of SPLVs (A) and MLVs (B). 4

Figure 4:

Differences in reduction of doxyl spin samples of SPLVs and MLVs by ascorbate.

Figure 5: ^S

Efficacy of two-step treatment of Brucella canis infection with streptomycin embedded in SPLV, as studied in the removed migratory spleen of B. canis infected mice. _£

Figure 6:

Effectiveness of a two-step treatment of B. canis infections using streptomycin embedded in SPLV, as studied in a removed organ of B. canis infected mice. ^£

Figure 7:

Efficacy of a two-step treatment of streptomycins incorporated into SPLV in guinea pigs infected with Brucella abortus. ⁶

858 2

Production of SPLVs

The SPLVs produced according to the present invention have unique properties that differ from liposomes known hitherto. The SPLVs' number is a lipid vesicle consisting of a few to about 100 bilayers.

The membrane bilayer is an amphipathic bpid bimolecular layers - which the non-polar hydrophobic hydrocarbon "tails" are oriented toward the center of the double TEG ³ - and the polar head ,, "out hydrophilic - comprises - that is pointing towards the aqueous phase. Laying the bilayers on top of each other, an aqueous compartment is formed which, on the one hand, is the size of the vesicle and, on the other hand, forms the area extending between the adjoining layers ®. Many proteins, glycoprotein, glycolipid, glucopolysaccharide, and other hydrophobic and / or amphipathic materials can be complexed with bpid bilayers.

SPLVs are prepared as follows: Va0 lamellae amphipathic lipid or mixture of lipids is dissolved in an organic solvent. Suitable for this purpose, but particularly preferred Mixtures of several organic solvents are diethyl ether, fluorinated hydrocarbons, and fluorinated hydrocarbons and ether ^5. To this solution is added the aqueous phase and the active ingredient to be incorporated. The resulting biphasic system is converted to SPLVs by emulsifying the aqueous material while evaporating the solvent. Evaporation of the solvent can be accomplished by any of the known evaporation techniques, such as gas vapor transfer through the mixture, heating, vacuum evaporation during or after ultrasonic treatment. The volume of the solvent used should be substantially greater than the volume of the aqueous phase in order to ensure complete emulsification of the aqueous material in the mixture. In practice, the process uses three volumes of solvent per volume of aqueous phase. In fact, the ratio of solvent to aqueous phase may be 100 or more per volume of aqueous phase. The amount of lipid should be described as sufficient, half present to cover the emulsified droplets (40 mg lipid / ml aqueous phase). In practice, the above ratios are influenced only by economy factors, but in practice, for 15 g of lipid, 1 ml of aqueous phase ⁰ results in SPLVs.

The bpid vesicles produced by the process of the present invention exhibit different supramolecular and non-conventional liposomes. In the process of the present invention, the temperature range used in the first step of the process is between 4 and 60 ° C, with respect to the transition temperature of the lipid phase used. The advantage of the above solution is that the denaturation of proteins in the temperature labile product is avoided since θ is incorporated into SPLVs, which can be produced from a phospholipid SPLVs such as distearoyl phosphatidylcholine, but converted to a conventional Eposome at a phase transition temperature. During the process, usually more than 20% encapsulated water soluble material and ⁵ more than 40% soluble EPID parent-41191 graphs. For MLVs, the amount of water phase incorporated is less than 10%.

The vast majority of amphipathic lipids can be components of SPLVs. Suitable hydrophilic groups include, but are not limited to, phosphate, carboxyl, sulfate, and amino groups. Suitable hydrophobic groups include, but are not limited to, saturated and unsaturated aliphatic hydrocarbon groups, wherein the aliphatic groups may be substituted with at least one aromatic and / or cycloaliphatic group. Preferred amphipathic compounds are phospholipids and molecules with closely related chemical structures. Such molecules include lecithin, phosphatidylethanolamine, lysolecithin, lysophatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, cardiolipin, phosphatidic acid and cerebrosides. Lipids useful in the preparation of SPLVs include phospholipids, including natural lecithins, (egg lecithin or soybean lecithin), and synthetic lecithins, such as saturated synthetic lecithins (e.g., dimyristoyl phosphate di lecithins (e.g., dioloylphosphatidylcholine or dilinoloylphosphatidylcholine). SPLV bilayers may contain a steroidal component such as cholesterol, compostanol, cholestanol, cholestane and the like. When compounds with an acidic hydrophilic group (phosphate group, sulfate group, etc.) are used, the resulting SPLV will be anionic; their base groups thus providing cationic hposomes using amino groups; the use of polyethyleneoxy or glycol groups results in neutral liposomes. The size of the SPLV varies within wide limits. This range is between 500 nm and 10,000 nm, generally between 1000 and 4000 nm.

Many biologically active compounds can be incorporated into SPLV (by incorporation into the aqueous portion or membrane bilayer). Such compounds include, but are not limited to, nucleic acids, polynucleotides, antibacterial compounds, antiviral compounds, antifungal agents, anti-parasitic compounds, antitumor compounds, proteins, toxins, enzymes, hormones, neurotransmitters, glycoproteins, immunoglobulins, immunomodulators, immunomodulators, immunomodulators, fluorescent substances, polysaccharides, cell receptor binding molecules, anti-inflammatory agents, agents for the treatment of glaucoma, mydriatic compounds, local anesthetics, etc.

For example, SPLV can be prepared by adding 50 micromoles of phospholipid to 5 ml of diethyl ether containing 5 micrograms BHT (butylated hydroxytoluene) and then adding 0.3 ml of an aqueous phase containing the biologically active substance to be encapsulated. The resulting solution, which contains the active ingredient and the incorporated lipid, is washed with ultrasound to remove most of the solvent with inert gas. So much- <

858 2 donations result in stable SPLV due to the incorporation of BHT into the vesicles.

Eur. J. Biochem. 121: 475-482 1982 (Leen et al.) Report the encapsulation of 5 liposomes of antibodies by sonication and evaporation by evaporation of the solvent from the chloroform / ether and aqueous phase systems containing cholesterol and phosphatidylcholine, but not details the amount of lipid remaining in the aqueous phase.

SPLV has distinctly different properties from other liposomes that contain one or more lamellae (e.g., SUV and RÉV). Fractional freeze electron microscopy studies show that SPLV formulations are substantially free of SUV, REV, i.e. less than 20% of the vesicles are unilamellar. However, these structures are indistinguishable from MLV by electromicroscopy, although they differ in many physical properties. The differences between SPLV and MLV are detailed below.

Storage stability of SPLV

The stability of the lipid vesicle shows how well the closed cavity of the vesicle is separated from the external environment over a prolonged period of time. A lipid vesicle can be used well if it is stable during storage. However, the slow release of the inner contents of the vesicle during use is also a very important factor for the application. In other aspects of application, it is essential that the vesicle remains intact after administration until the active site is reached. Our experiments show that SPLV meets these requirements, but MLV does not.

The decomposition of the vesicles is caused by two factors. One such factor is the auto-oxidation of lipids, whereby their hydrocarbon chains form peroxides which render the bilayer unstable. This oxidation can be immediately eliminated by the addition of antioxidants such as butylated hydroxytoluene (BHT). The decomposition of the vesicles can be caused by an agent in the environment that disrupts the bilayer structure of the lipids and creates a pore on the same membrane.

Lipid vesicule formulations are initially white in color. Auto-oxidation causes the preparation to discolour (tan). Comparison of MLV and SPLV formulations shows that although the same lipid and aqueous components are used in their preparation, MLV becomes discolored within two weeks, whereas SPLV remains white for at least two months. These observations are also supported by thin layer chromatography, which indicates that lipid degradation products are not derived from SPLV but from MLV. In the event that both formulations are administered 5

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Treated with BHT, MLV will slowly but discolor within one month, while SPLV will remain white and stable for at least 6 months, but in many cases longer.

Table I shows that antibiotic-containing SPLV formulations remain stable for more than 4 months when placed in neutral pH η saline buffer. These results indicate that none of the antibiotics encapsulated in SPLV is degraded or degraded during the study.

Other studies have shown that SPLV is also capable of enclosing or isolating other molecules, such as small ions such as calcium. Arsenic III is a dye that changes its color from red to blue in the presence of divalent calcium ions. The stability of the vesicles can be measured by observing the color change of the system by encapsulating the dye in SPLV and placing it in a calcium chloride buffer. No change from the original color was observed after 6.5 months, indicating that neither the dye nor the ion was released from the microcapsule.

Table I

Egg phosphatidylcholine SPLV stability in a closed container at 4 ° C after 4.5 months ³

built-in agent Initial Emptying of Integration Super- The built-in agent bioavailability % % watering ⁰ streptomycin 34.1 0 97 sulfate spectinomycin 37.2 0 84 chloramphenicol 35.2 0 89 oxytetracycline 18.8 0 91 erythromycin 0.4 0 97 Sulfamerazine 6.3 0 93

a) SPLV was prepared using 127 micromoles of egg phosphatidylcholine (EPC) and 25 micromoles of active ingredient.

After storage for 4.5 months at 4 ° C, the SPLV was removed from the storage buffer by centrifugation. A series of dilutions were prepared from various amounts of SPLV and the supernatant was bacterially assayed for bioactivity relative to standard antibiotic dilution.

b) 0 indicates that we are below the measurement limit

The above experiments show that SPLV is very stable during storage and use. The production of MLV which appears to be stable for such a long period of time, but in this case requires the use of synthetic lipids such as DSPC, makes the process very costly and practically unworkable.

Stability of SPLV in other media

A method of examining the molecular arrangement is accomplished by placing the lipid vesiculu5 moiety in a medium containing membrane perturbing agents. Depending on the membrane structure, different vesicles behave differently to different agents.

During the experiments, vesicles containing a radiolabeled molecule ( ³ H inulin) in a closed aqueous portion were prepared. Inulin polysaccharide, which is incorporated into the aqueous phase when radioactively labeled, is capable of monitoring the aqueous portion of lipid vesicles. After treatment with such an agent for a period of time, the vesicles were separated from the medium by centrifugation and the relative radioactive amount removed from the vesicles (into the medium) was measured. II. Table 20 summarizes these results; the values represent the percentage of decay showing the distribution of the radioactive material in the surrounding medium relative to the initial vesicle.

II. spreadsheet

Stability of SPLV in other media

Incubation medium ³ Decomposition% MLV SPLV Hydrochloric acid 0.125M 90.5 55.2 Urea _1M 21.7 44.8 guanidine 0.5M 5.7 7.4 LOM 8.3 10.1 Ammonium acetate 0.5M 27.0 67.0 l, 0M 25.9 54.7 to 63.1 Serum 76.2 57.8

a The incubation time is 2 to 4 hours or 1 hour for hydrochloric acid.

The SPLV hydrochloric the MLV is substantially more stable than ⁵ θ. II. Table IV shows that, like MLV and SPLV, egg lecithin is destabilized within one hour by treatment with hydrochloric acid. However, it should be noted that SPLV is less sensitive to acid treatment than MLV. It is believed that these results essentially reflect the actual interaction between the lipids and their environment.

Treatment with SPLV with urea differs from MLV (Figure 1, Table II). The ®0 urea has a chaotropic effect and a strong dipole moment. It has been observed that SPLV is much more sensitive to urea treatment when it contains an osmotic agent such as sodium chloride at the same ⁶⁵ concentrations. (Figure 1). MLV is urea

-61 is not significantly decomposed in the medium to a value greater than that present in the medium. Although the explanation for this phenomenon is theoretically, the answer seems to be due to the dipole effect, and not so much the chaotropic property, because guanidine, a structure similar to urea, does not destabilize SPLV (Table II). Although guanidine is strongly chaotropic, it has no strong dipole moment.

SPLV also appears to be sensitive to ammonium acetate, whereas MLV does not.

(Table II). However, neither ammonium ion (ammonium chloride) nor acetate ion (sodium acetate) cause destabilization of SPLV. It seems that the polarity of ammonium acetate, rather than the ionic nucleus, is responsible for inducing decay.

Initially, these results seemed surprising because SPLV is much more stable than MLV when incubated in your blood, such as serum or blood. However, these assumptions are theoretical and, of course, other explanations are possible. If the stability of SPLV is due to the unique structure of membrane bilayers, that is, the polar groups of membrane lipids are hydrated by clouds of oriented water molecules or enclosed in a hydrate envelope, many molecules are likely to break or interact with this hydrate envelope. changes in the integrity of the membrane structure and thus decay.

Regardless of theoretical considerations, SPLV is destabilized in urea, and the results demonstrate that there are distinct differences in structure between MLV and SPLV. These differences are very well suited for use. As mentioned above, SPLV decomposes very slowly during eye treatment. It is believed that this desired slow release of drug is similar to the destabilization of SPLV in tear fluid.

In serum, SPLV is more stable than MLV. Lipid vesicles are often used intraperitoneally for use in the treatment of brucellosis. In order to achieve the desired effect, the vesicles must be stable for a sufficient period of time until the target cell or tissue is reached. SPLV and MLV prepared from egg lecithin were reacted with bovine embryo serum containing the active ingredients (Table II). After 48 hours at 37 ° C, SPLV was found to be more stable than MLV.

Characteristics of SPLV in vivo dosing

In therapeutic systems, in vivo, SPLV is particularly suitable for use as a carrier because of its many characteristics.

(A) SPLV is resistant to cleaning. When used to treat an organism with SPLV, they are all components of lipids

I 858 2 both retained the active ingredient in tissues and cells.

(B) They also form a sustained release system for the production of SPLV. During dosing, the

SPLV is stable and also appears to be stable during storage and in the presence of body fluids, but with slow in vivo delivery, the active ingredient is slowly dissolved to provide a sustained action.

(C) Due to the high degree of incorporation and high stability, an effective dose of the active ingredient is released during treatment.

(D) Preparation of SPLV is very economical because the stability of the vesicles eliminates the need to incorporate expensive stabilizers into the bilayers.

The following experiments illustrate some of the above features of SPLV when applied locally to 20 eyes of test animals. The SPLV used in the experiments was prepared as described above with the exception that the lipid bilayers and the active ingredient were radiolabelled in order to monitor these components in the ocular tissues over a period of time.

SPLV was prepared from 100 mg egg phosphatidylcholine (EPC) and 100 mg gentamicin sulfate. The lipid component was radiolabelled by incorporating ^12S J-phosphatidylethanolamine ( ^12S J = PE) in pressurized amounts in the bilayers and the drug was labeled with ¹²⁵ J-gentamicin sulfate ( ^I2S JGS) in the aqueous phase. The SPLV was rinsed several times with buffer to effectively remove material that was not incorporated or encapsulated.

An aliquot of the SPLV preparation was removed and extracted to separate the aqueous phase from the organic phase. The radioactivity of each phase was measured and the initial ratio of ¹²⁵ J-PE: ¹²⁵ J-GS [cpm (bpm / min) in the lipid phase: cpm in the water phase] incorporated in SPLV was determined.

Extraction was carried out as follows: 0.8 ml of a 0.4 M aqueous sodium chloride solution, 1 ml of chloroform and 2 ml of methyl alcohol were stirred until a homogeneous phase was obtained. To the mixture was then added 4 microliters of radiolabeled SPLV. Upon dissolution of the SPLV in the organic phase and in the aqueous phase, the reaction mixture, which was initially cloudy, became clear. The phases were separated by addition and mixing with 1 ml of 0.4 M aqueous sodium chloride and 1 ml of chloroform and centrifuged at 2,800 x g / 5 min. One ml of each phase was withdrawn and the radioactivity (in cpm) was measured. (Initial rate:

¹²⁵ J-PE: ^12s J-GS = 1.55: 1.)

Fifteen Swiss Webster female mice were treated and immobilized with an analgesic to prevent eye injury. An aliquot of 2 µl aliquots of the radiolabeled SPLV suspension was administered topically to each eye. Groups of three animals were sacrificed at 1, 2, 3, 18 and 24 hours. Nine

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III. Table III shows the same treatment of female Swiss Webster mice (control group), except that an aliquot of 2 μΐ of an aqueous solution of radiolabeled gentamicin sulfate was applied locally to each eye. Control animals of three groups were sacrificed after 1.4 and 8 hours. After killing the animals, the eyelids were removed immediately, minced, and extracted using the procedure described above to separate the aqueous components from the lipid components. The radioactivity of the phases was determined (total number of radioactive readings were counted). The radioactivity measured in the lipid phase shows the retention of SPLV lipids in the ocular tissue, while the radioactivity in the aqueous phase shows the retention of gentamicin in the ocular tissue. Figure 2 graphically illustrates the retention of each component in the eyelid tissue (expressed as a percentage of the original cpm).

Figure 2 clearly illustrates the retention of the SPLV Epid component in the eyelid tissue over a 24 hour period and the sustained release of gentamicin from the SPLV over a 24 hour period (expressed as a percentage of gentamicin remaining in the eyelid tissue during this time). shows the rapid elimination of unencapsulated gentamicin (locally administered aqueous gentamycin) from the eyelid tissue, for example, the control gentamicin solution was cleared from the ocular tissue within 4 hours (less than 5% of the amount of gentamicin remaining in the eyelid tissue). more than 50% remained in ocular tissue during this 4-hour period, and more than 15% of SPLV-encapsulated gentamicin remained in the eyelid after 24 hours, which indicates that nearly 85% of SPLV-encapsulated gentamicin after 1 hour, while the unencapsulated gentamicin was dissolved 95% of the tree was removed within a four-hour period.

Table 3 compares the proportion of SPLV Epid Phase: Aqueous Phase in Eyelid Tissue at each time point. An increase in this ratio indicates the release of gentamicin from SPLV.

The bioactivity of SPLV encapsulated gentamicin sulfate in the eyelid tissue was also evaluated. Gentamicin sulfate was recovered by extraction of the aqueous phase of the eyelid three hours after treatment with SPLV-encapsulated gentamicin sulfate. A dilution series of the aqueous phase was prepared and 2 aliquots of this were mixed with Staphylococcus aureus on an agar plate; After 24 hours of incubation, inhibition zones were measured. After extraction of the ocular tissues of the animals treated with SPLV-encapsulated gentamicin sulfate, gentamicin sulfate was recovered and it was determined that its bioactivity was completely preserved.

Prolonged release of SPLV-encapsulated gentamicin after topical application in the whole eye

Time Aftercare Total amount of SPLV recovered from the eyelid % of the initial dose expressed Lipid: aqueous phase ratio of SPLV in the eyelid ( ^12S J-PE: ¹²⁵ J: GS 0 100% 1.55 1 hour 100% 2.1 ϊ hours 100% 2.82 18 hours 94% 6.89 24 hours 85.1% 7.17

Electron spin resonance studies

Although SPLV and MLV appear to be identifiable by electromicroscopy, 25 ESR (electron spin resonance) spectroscopy shows very precisely the differences between their supramolecular structures. The SPLV MLV TOI distinguished on the basis of molecular structure, providing evidence of an error megnöveke ³⁰ molecular orbital, increased molecular motion and greater penetrabiEtását ascorbate. These differences in the structure of the molecule determine the various biological effects.

In the electron spin resonance spectroscopic studies of spin probe ³⁵ using 5-doxyl stearate (5DS) was incorporated into the EPID bilayer. The unmatched electron of the doxyl group absorbs microwave energy as the sample is placed in the magnetic field. From the absorption spectrum, ⁴⁰ empirical parameters can be determined: S, the path parameter; _{O is} the hyperfine binding constant and Tau is the rotational correlation time. Figure 3 illustrates a typical signal where A is the SPLV signal and B is applied to the MLV signal, both with 5-doxyl stearate signal ⁴⁵ comment. The spectrum was recorded at 100 Gauss scan range at room temperature. The track parameter (s) that are both 2Ti and 2T !! dependent, the deviation of the observed ESR signal from the fully uniformly oriented probe is measured.

⁵⁰ For a uniformly oriented sample, S = 1.00, for a disordered sample, S is 0. The hyperfine binding constant _{o o is} calculated from the values of 2T) and 2Tj j, and shows the local polarity, which indicates the position of the spin probe on the membrane within. The rotational correlation time (which W _o, h _o depends, hT value) means the time during which the molecule is "forget" térbeE previous orientation. The IV. Table 5 shows the typical ESR ⁶⁰ differences between SPLV and MLV with a 5-DS spin probe.

191,858

Although in both cases the spin probe gives the same depth of the bilayer, SPLV has a significantly higher molecular path and degree of molecular motion than MLV.

The differences between SPLV and MLV are illustrated by the ability to ascorbate, which reduces the doxyl spin probe. It has long been a well-known fact that ascorbate reduces doxyl levels, presumably like its hydroxylamine analogue, which does not absorb microwave energy in a magnetic field. In aqueous solution, the reduction occurs rapidly with the disappearance of the accompanying ESR signal. If the spin probe is located in a protected environment, such as a lipid bilayer, it is not reduced much more slowly or at all by hydrophilic ascorbates. Accordingly, nitroxide reduction can be used to study the rate of penetration of ascorbate into lipid bilayers. Figure 3 shows the% spin turn time of the ascorbate solution suspension of SPLV and MLV. After 90 minutes, the ascorbate of the MLV probe was reduced to 25%, but 60% of the probe was inserted into the SPLV. SPLV allows significantly higher ascorbate penetration than MLV.

ARC. spreadsheet

Characteristics of SPLV and MLV ESR

Tau s ^A o SPLV 2.65 x 10 ^_ 9 seconds .614 14.9 MLV 3.65x10sec .595 14.9

VI. spreadsheet

Incorporation of active substance into SPLV

The outstanding advantage of SPLV over conventional MLV is that it is capable of capturing and preserving a greater percentage of an active ingredient (see Table V).

Table V.

Comparison of MLV and SPLV

Expressed as% of material incorporated

MLV SPLV Encapsulated material: inulin (in water) 2-6% 20-30% bovine serum albumin 15% 20-50% streptomycin 12-15% 20-40% polyvinylpyrrolidine (in the water) 5% 25-35%

Interaction of SPLV with cells

Another unique feature of SPLV is that it interacts with cells so that a relatively greater amount of encapsulated material contained in the vesicles is dispersed through the cytoplasm of the cells, which is greater than the limit value of the phagocytic vesicles. When mixed with SPLV cells, the two substances combine. The fusion is effected such that SPLV, in contrast to MLV, interacts with cells in vivo, such that each cell contains at least some of the substances originally incorporated into SPLV. This substance is distributed between cells and is not limited by phagocytic vesicles. This phenomenon can be demonstrated by the incorporation of ferritin into the aqueous phase of a SPLV formulation. After 20 cell fusion, fine structure analysis shows that ferritin is distributed through the cytosol and does not bind to intracellular membranes. This phenomenon can also be detected in MLV, but SPLV provides transfer of a larger amount of the given substance.

Density of SPLV

The density of SPLV is much lower than that of MLV. VI. Table 3 shows the values measured by the ficol gradient stratification.

Density MLV SPLV 40 ficol top coat 1% top coat 0,5% gms / ml 1,071 1.0274

Volume of SPLV

Collected by centrifugation at 1,000-100,000 x g, the SPLV forms a sphere with the same phospholipid concentration as MLV, but is significantly larger in size. 50 At 16,000 x g, the resulting SPLV sphere is approximately one-third the size of MLV.

Osmotic properties of SPLV

Because the phospholipid bilayers are permeable to water, placing the MLV in a hypertonic environment results in an osmotic force on the entrapped water. SPLV contracted more than MLV. After 16 hours of buffering, the internal salt60 concentration is 20 times higher, and SPLV does not contract to the same final volume as MLV. (SPLV spheres remain one-third larger than MLV spheres.) This indicates that the spherical size differences are not due to the volume differences of the enclosed water.

-9191 858 2

Use of SPLV

SPLV is particularly useful in systems where the following factors are of particular importance: storage and stability in contact with body fluids; a relatively high degree of encapsulation; economy; release of the incorporated material in a biologically active form.

Depending on the mode of in vivo administration, SPLV is resistant to rapid dissolution (very significant in sustained release therapeutic systems) or penetrates into RES cells.

The SPLV produced by the process of the invention can be used in various systems. SPLV can be used to enhance therapeutic efficacy of therapies, treat infections, enhance enzyme replacement, oral drug delivery, topical drug delivery, in vitro and in vivo genetic information into the cell, vaccines, recombinant DNA segments, cellular diagnostics, for use in which the incorporated "re port er" molecule is dissolved. SPLV is also an encapsulation agent for cosmetic formulations, pesticides, and substances that can influence the growth of the plant with sustained release formulations.

The terms related to the use of SPLV used in the following assays are identical in functional terms to SPLV or any other liposome or lipid germ sugar.

Dissolution of biologically active compounds

Under in vitro conditions, delivery of the active ingredients to the cell (e.g., animal cells, plant cells, protozoa, etc.) requires that the SPLV containing the active ingredient be added to cell culture. However, SPLV is also capable of delivering the drug in vivo (in animals, humans, plants and protozoa). Depending on the purpose of administration, SPLV can be administered in a variety of ways, including, but not limited to, human and animal administration; injection by intravenous, intraperitoneal, intramuscular, subcutaneous, intraauricular, intramuscular, intraurethral, etc. It happens. SPLV can also be administered topically (e.g., on a damaged surface). SPLV is absorbed through the epithelium or mucous membranes, such as ocular epithelium, oral mucosa, rectal and vaginal epithelial fibrils, respiratory tract fibrils, nasopharyngeal mucosa, intestinal mucosa, etc.). In plants and protozoa, direct treatment can optionally be achieved by dispersing in the natural medium of the organism to be treated, or by adding it to the surrounding medium or to water.

The route of administration is also determined by the location of the cells of the organism to be treated, where the active ingredient is to be delivered. Accordingly, for example, in a specifically infected area, topical treatment is most convenient (if the infection is extreme). Delivery to the circulatory system (reticular loendothelial cells) is most easily accomplished by intravenous, intraperitoneal, intramuscular, or subcutaneous injection.

Because SPLV provides a sustained release, once or more hairs may be administered once toxic. The following describes some systems suitable for general use of SPLV, without limiting our invention thereto.

Treatment of diseases

Many human, animal and plant diseases can be treated more effectively than before with SPLV containing the appropriate compound or compounds in encapsulated form. Infections (intracellular and extracellular, cysts, tumors and tumor cells, various allergies, etc.) can be well treated with the SPLV of the invention.

There are many general principles that can be used to mediate with SPLV; In many systems, the very advantageous feature of SPLV that it combines with macrophages when administered in vivo is particularly useful.

In the case of an intracellular infection, SPLV can be used as a therapeutic system to deliver the site of infection. Such infections include infections of cells of the reticuloendothelial system such as brucellosis. These intracellular fer35 infections are very difficult to treat for the following reasons: 1. Because the infection is within the cells of the reticuloendothelial system, no therapeutic agent is introduced into the circulation because it does not penetrate the cell membrane at a concentration corresponding to a therapeutic amount; very difficult to handle; 2. In many cases, the administration of therapeutic eyes only in toxic amounts is sufficient to treat such infections; 3. the treatment must be fully effective in such a way as to prevent re-infection or spread of the infection to other individuals after treatment.

SPLV containing the appropriate biologically active agent (dosing preferably 50 i.p. is intraperitoneal or intravenous) may be used if the individuals to be treated are bacilli or potential bacilli, such as infected and uninfected animals in animal herds.

Because phagocyte cells combine with SPLV, a biologically active substance encapsulated in SPLV contacts the infecting organism at the site of infection. Thus, the method of the invention optionally infection caused by several different kinds of microorganisms for ⁶⁰ avoid. Examples of microorganisms, bacteria, parasites, fungi, mycoplasmas and viruses such as Brucelia spp., Mycobacterium spp., Salmonelle spp., Listeria spp., Francisella ⁶⁵ spp., Histoplasma spp., Corynebacterium spp.

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Coccidiodes spp. and infections caused by lymphocyte choriomeningitis virus.

The therapeutic agent is selected according to the infectious microorganism. For example, bacterial infections can be treated with an encapsulated antibiotic. The antibiotic is in the aqueous phase of SPLV and / or is incorporated into the bilayer of the vesicle. The following antibiotics may be used in the process of the present invention, but are not limited to these; penicillin, ampicillin, hetacillin, carbencillin, tetracycline, tetracycline hydrochloride, oxytetracycline, hydrochloride, chlorotetracycline hydrochloride,

7-chloro-6-dimethyltetracycline, doxycycline monohydrate, methacycline hydrochloride, minocycline hydrochloride, rolitetracycline, dihydrosstreptomycin, streptomycin, gentamycin, kanamycin, neomycin, erythromycin, carbomycin, oleandomycin, oleandomycin, oleandomycin, oleandomycin, dihydrate, and cephalexin ² monohydrate.

Effective methods for treating brucellosis are described herein (see Examples, infra.) The efficacy and efficacy of the composition of the invention is provided. Surprisingly, it has been found that the therapeutic system in question is also capable of treating infections for which treatment with an antibiotic incorporated in MLV has been shown to be ineffective. Effective treatment was not predictable as residual infections were dispersed and the cycle of infection was expected to recur. The composition has also been successfully used to treat lymphocyte choriomeningitis virus infection. *

Of course, the SPLV produced by the process of the present invention is not only useful for treating intracellular infections. SPLV can also be delivered to intracellular and extracellular areas of infection. For example makrofágo- ^z salts can be used to deliver the active substance in a system to a point of extracellular infections. Accordingly, SPLV is used to deliver a therapeutic agent to uninfected macrophages by administering SPLV under in vivo conditions, preferably intraperitoneally or intravenously.

Macrophages the SPLV united with the therapeutic agent and loaded ,, approximately ", generally speaking, the equine ^£ macrophages bio logically active agents include maintaining for about 3-5 days. When the "loaded" macrophages reach the site of infection, the pathogen fuses with the macrophages. As a result, the pathogen, the therapeutic agent ^{of this} expressly included Zo united macrophage and so decomposed. In this way, particularly human and bovine Staphylococcus aureus mastitis can be treated.

If the pathogen infection or other external influences or accessible location, the built ^(SPLV into therapeutic agent is applied locally. Particularly preferably be treated in case of injury to the eyes. Eye lesions SPLV containing one or more appropriate active ingredients may be locally damaged face. Szemfertőzé- ⁶

858 2 microorganisms can be caused by animals and humans. Such microorganisms include, but are not limited to, Moraxella spp., Clostridium spp., Corynebacterium spp., Diplococcus spp., Flavobacterium spp., Hemophilus spp., Klebsiella spp., Leptospira spp., Mycobacterium spp., Neisseria spp. spp., Proteus spp., Pseudomonas spp., Serratia spp., Escherichia spp., Staphylococcus spp., Streptococcus spp. and bacterial-like microorganisms, including Mycoplasma spp. and Rickettsia spp. These infections are difficult to treat with conventional methods, as infected areas remain after treatment, which causes re-infection due to tearing. Also described herein is the treatment of eye infections caused by Moraxella bovis with SPLV (see examples, infra)

Because SPLV is resistant to fission and is able to release its contents over a long period of time, SPLV is also capable of treating a number of injuries that require the sustained action of the active ingredient at the site. For example, glaucoma is characterized by an abnormal gradual increase in intraocular intraocular pressure, which causes a gradual loss of peripheral vision and, in the absence of medical intervention, a loss of central vision, leading to blindness. The drugs used to treat glaucoma today may optionally be administered topically as eye drops. In many cases, however, drips are required every 15 minutes because the drug is rapidly removed from the eye. If an eye disorder such as glaucoma is treated with a composition of the invention, i.e., pilocarpine, Floropryl, physostigmine, carcholine, acetazolamide, ethazolamide, dichlorophenamide, carbacho, demecarium bromide, diisopropyl phospho date, ecothioplat iodide, physostigmine or neostigmine, etc. Built into SPLV, the resulting therapeutic system can be successfully used to treat the patient's eye.

Without limiting our need, other substances encapsulated in SPLV can be used locally; thus, mammary dilating agents (e.g., epinephrine, phenylepinephrine, hydroxy amphetamine, ephedrine, atropine, homatropine, scopolamine, cyclopentolate, tro picamide, encatropine, etc.); topical anesthetics, antiviral agents (e.g., iodo-uridine, adenine-arabinoside, etc.); antimycotic agents (e.g., amphoteracin B, natamycin, pimaricin, flucytosine, nystantine, thimerosal, sulfamerazine, thiobendazole, tolnaftate, griseofulvin, etc.); anti-parasitic agents (e.g., sulfonamides, pyrimethamine, clindamycin, etc.); and anti-inflammatory agents (e.g., corticosteroids such as ACTH, hydro-cortisone, prednisone, medrysone, beta-methasone, dexamethasone, fluoromethalone, triamcinolone, etc.).

The invention is illustrated by the following examples, without, however, limiting it.

-11191,858

Production of SPLV

As described above, during the preparation of SPLV, a lipid or mixture thereof is dissolved in an organic solvent, an aqueous solution of the substance to be encapsulated is added to the solution, and the mixture is sonicated. Preferably, the solvent is removed during the ultrasonic treatment, but the organic solvent can be removed by any evaporation technique both during and after the sonication. The SPLV described in the present invention is prepared as follows (any other method may be used).

Preparation of antibiotic-containing SPLV

Lecithin (100 mg) was dissolved in diethyl ether (5 ml). The reaction mixture was placed in a round-bottomed flask. To a solution of the lipid in diethyl ether was then added at room temperature 100 mg of streptomycin sulfate in 5 mM HEPES (4- [2-hydroxyethyl] piperazino 2-ethane sulfonic acid) 0.0725 mol NaCl / 0.0725 mol KCl pH 7.4, A solution of 3 ml was added. The reaction mixture was then emulsified in an ultrasonic bath (Laboratory Supplies Co., Inc.) (Model 10536) for several minutes (80 KkHz frequency: 80 watts power output) and simultaneously dried under a stream of nitrogen to a viscous paste.

To the remaining viscous paste was added 10 mL of 5 mM HEPES. The resulting SPLV formulation, which contains streptomycin, was suspended in a buffer solution, shaken for several minutes in a vortex mixer, and centrifuged (12,000 x g, 10 minutes, 20 ° C) to remove non-encapsulated streptomycin. The resulting cake was suspended in 0.5 mL of 5 mM HEPES.

Dihydrostreptomycin, or gentamicin sulfate, or ampicillin, or tetracycline hydrochloride, or kanamycin-containing SPLVs are prepared as described above.

Preparation of SPLV containing other membrane components

The procedure described above, except that one of the following substances is mixed with egg lecithin: 1. the phosphatidic acid gives an 8: 2 molar ratio of lecithin: dicetylphosphate; 2. stearylamine results in an 8: 2 lecithin: stearylamine composition; cholesterol and stearylamine result in a 7: 2: 1 lecithin: cholesterol: stearylamine composition;

Production of SPLV with and without BHT

Butylhydroxytoluene (BHT), 1 μg / ml, was added to the distilled ether for storage. Proceeding as described above, the un distilled ether is used as a solvent for incorporation of BHT into SPLV.

SPLV without BHT4 was prepared as described above using distilled ether as solvent.

SPLV release in vitro.

The antibiotic delivery to SPLV-mediated macrophages in culture media is shown below.

Peritoneal macrophages were prepared by peritoneal lavage in C ₅₇ BLK adult male mice, and the macrophages were resuspended in minimal medium (MEM) pH 7.2 at 10% heat inactivated bovine embryo serum. The cell suspension was adjusted to 1 χ tissue culture 10 ⁶ cells / ml. Media containing peritoneal macrophages at a concentration of 1 χ B. canis were added to 10 ⁶ CFU (colonyformingunits) / ml. After 12 hours, bacteria that were not covered by peritoneal macrophages were removed by repeated washing. After washing the peritoneal macrophage culture, the medium was divided into five sections such that each group contained 12 replicate cultures. Each group was used as a control which was not treated. Group 2 was treated with aqueous streptomycin sulfate at a concentration of 1 mg / ml. Buffer saturated SPLV was added to group three. Group four included 1 mg / ml streptomycin sulfate and buffer saturated SPLV. Group five represents SPLV containing 1 mg / ml streptomycin sulfate. After 24 hours, the supernatants were removed by repeated washing and the peritoneal macrophages were disrupted by repeated freezing and thawing. The dilution series of the macrophages disrupted was plated on brucella agar and, after 4 days, the amount of surviving B. canis was determined from the dilution series. The results obtained are shown in Table VII. Table 1 shows that streptomycin incorporated into SPLV completely abolishes intracellular B. canis infections under in vitro conditions.

The above experiment was also performed for B. abortus except that peritoneal macrophages were prepared by peritoneal lavage of adult female albino guinea pigs. The results obtained are shown in Table VII. is summarized in Table.

Preparation of SPLV containing Pilocarpine

The procedure described above, except that pilocarpine is used in place of the antibiotic.

-12191858

VII. spreadsheet

Colonel units of intracellular Brucella isolates after treatment with SPLV-infected macrophages containing streptomycin

B. canis ³ B. abortus ^ control 2.6 ± 1.13x10 ³ 0.81xl0 ± 3.1 * Buffer-saturated SPLV 2.82 * Ό.10χ10 ³ 2.9 * ± 0.17xl0 Free streptomycin 3.11 + 0.40xl0 ³ 3.3 ± 0.25x10 * Streptomycin ⁺ Buffer-saturated SPLV ^C 2.76 + 0.20xl0 ³ 2.8 * ± 0.42xl0 Streptomycin ⁰ incorporated into SPLV 0 0

colony forming units (CFU) for B. canis (12 replicates). The isolates were obtained from peritoneal macrophages of pre-infected mice (C _s7 Blk).

b Peritoneal macrophages of the same number of pre-infected guinea pigs. B. abortus CFU (12 replicates).

c Streptomycin concentration 1 mg / ml.

Treatment of intracellular infections

The use of SPLV in the treatment of intracellular infections is described below. The data obtained show: 1. the efficacy of the antibiotic encapsulated in SPLV in therapy; 2. the multiplication of the SPLV formulation with increased efficacy. Comparison of the MLV and SPLV used as the vehicle is described in the protocol.

Brucellosis causes serious economic and public health problems worldwide. Brucellosis is a disease of Brucella spp. causes. Brucellosis can spread to many mammals, including humans, domestic animals and various wild animals. The following six Brucella spp. induces brucellosis in animals: B. abortus, B. canis, B. melitensis, B. neotomae, B. ovis and B. Suis. Both domestic and wild animals can be potential bacilli hosts for brucellosis,

These infections cannot be treated with antibiotics because the infectious microorganism stays within the cell's reticuloendothelial system and is resistant to the bactericidal effect of the antibiotic. The dose level of antibiotic and the duration of treatment result in toxic effects and undesirable high levels of antibiotics in animal tissues. A further difficulty in the therapy of the above diseases is the residual infection (which is dispersed) and the cycle of infection. The economic disadvantage of such diseases can be estimated at millions of dollars due to spontaneous abortions in cattle breeding.

The incorporation of an antibiotic into SPLV is described below, followed by treatment with the encapsulated active ingredient in the intraperitoneal treatment of infected animals.

Treatment of B. canis infection with an antibiotic incorporated into SPLV

Eighty adult male Swiss mice were challenged intraperitoneally (IP) with B. canis ATCC 23365 (1 x 10 ⁷ CFU) and eight groups were formed, each containing ten mice. 7 days after inoculation with B. canis, the groups were treated as follows: Group 1 - no treatment, control; Group 2 - treated with buffer-saturated SPLV (0.2 mL IP); Group 3 - treated with aqueous streptomycin sulfate (1 mg / kg body weight, total volume: 0.2 mL IP); Group 4 - aqueous streptomycin sulfate (5 mg / kg body weight) total volume: 0.2 ml IP: Group 5 - aqueous streptomycin sulfate (10 mg / kg body weight) total 0.2 ml IP; Group 6 - Total volume treated with SPLV (1 mg / kg body weight) containing streptomycin sulfate: 0.2 ml IP;

Group 7 - Streptomy cin sulfate containing SPLV (5 mg / kg body weight) total volume: 0.2 ml I.P. Group 8 - Total volume treated with SPLV containing streptomycin sulfate (10 mg / kg body weight): 0.2 ml I.P. 14 days after inoculation with B. canis, all animals were sacrificed and the spleen was removed aseptically. The spleen was homogenized, serially diluted, transferred to brucella agar, and the number of surviving B. canis in the spleen after treatment was determined. The results obtained after four days of incubation are shown in Table VIII. Table.

VIII. spreadsheet

Efficacy of different concentrations of free or SPLV-encapsulated streptomy cin in mice infected with B. canis

Colony forming units B. canis / step

control Untreated 3.46x10 ^and ± 2.7x10 ⁶ Buffer-saturated SPLV 4.1xl0 ⁶ ± 0.66xl0 ⁶ Streptomycin- Free Locked in SPLV concentration streptomycin streptomycin (mgkg body weight) 1 1.5 ± 0.45x10 ⁶ 1.01 ± 0.25xl0 ³ 5 2.12 ± 1.71xl0 ⁵ 1.52 + 0.131x10 ⁴ 10 9.66 + 3.68x10 * 1:32 ± 1.00xl0 *

(a) I.P. total injection volume: 0.2 ml (sterile saline)

b) Amount of surviving B. Canis was isolated spleen (CFU value and 10 animals ± S.D.) expressed as experiment ratio (triplicate repeated).

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Effectiveness of treating multiple cancers of B. canis using antibiotics built into SPLV.

Eighty adult male Swiss mice were infected with B. canis ATCC 23365 (1 × 10 ⁷ CFU, IP) and eight experimental groups each containing ten mice were established. Seven and ten days after inoculation with B. canis, the groups were treated as follows: Group 1 - untreated, control; Group 2 - Buffer-saturated SPLV (0.2 ml,

I.P.); Group 3 - Whole dose treated with aqueous streptomy cin sulfate (1 mg / kg body weight) 0.2 ml I.P. Group 4 - A total dose of 0.2 ml I.P. treated with aqueous streptomy cin sulfate (5 mg / kg body weight); Group 5

- a total dose of 0.2 ml I.P. treated with aqueous streptomy cin sulfate (10 mg / kg body weight); Group 6 - total dose of 0.2 ml I.P. treated with 1 mg / kg body weight streptomycin sulfate in SPLV. Group 7 - total dose of 0.2 ml I.P. treated with 5 mg / kg body weight streptomycin sulfate in SPLV. Group 8 - Total dose of 0.2 ml I.P. treated with SPLV encapsulated streptomycin sulfate (10 mg / kg body weight). 14 days after inoculation with B. canis, all animals were sacrificed and the spleen was removed aseptically. The spleen was then homogenized, serially diluted, plated on brucella agar, and the number of B. canis surviving in spleen per treatment was determined. The results obtained after four days of incubation are shown in Figure 5.

The results of two-step in vivo treatment of B. canis infections are summarized in Figure 5. The results show that in the groups receiving the animals with aqueous streptomycin, the reduction in surviving B. canis in the spleen was only very small 7-10 days after inoculation. It was only in the streptomy cin-treated groups (7 and 10 days after incubation) that the bacteria were eliminated from the spleen of infected animals only in groups treated with 10 mg / kg SPLV.

In addition to the experiments described above, various tissues of mice infected with B. canis were treated twice with infection with streptomy zinc encapsulated in SPLV, and the following results were obtained and assays were performed as follows.

Thirty adult male Swiss mice were inoculated with B. canis ATCC 23365 (Ix10 ⁷ CFU, IP). Seven days after inoculation, the animals were divided into three groups with ten animals per group. Group 1

- control, no treatment; Group 2 - (7 and 10 days inoculation) dose of aqueous streptomycin sulfate (10 mg / kg body weight): 0.2 ml I.P. Group 3 - (Inoculation for 7 and 10 days) Dose volume treated with SPLV encapsulated streptomycin sulfate (10 mg / kg body weight): 0.2 ml I.P. 14 to 75 days after inoculation with canis, all animals were sacrificed and the organs described below were aseptically removed, homogenized, and serially diluted in

B. canis was isolated on brucella agar: heart, lungs, spleen, liver, kidneys, testes. Fig. 6 shows the surviving B. canis results for each organ after four days of incubation.

Mice infected with B. canis were treated twice with streptomy cin and the results of tissue examinations in tissues are shown in Figure 6. The results show that all 10 tissue samples of animals treated with streptomy cinn in SPLV were completely free of B. canis 14-75 days after inoculation with B. canis. Comparison of the results obtained with animals treated (or untreated) with aqueous streptomy cin at the same concentration and in the same mode of administration and animals treated with streptomycin incorporated in the same concentration and manner in SPLV showed that in the latter case after inoculation with B. canis 14-75 days in no tissue

B. canis isolated.

Comparison of the effectiveness of treatments with MLV and SPLV

Fifteen adult male Swiss mice were inoculated with B. canis ATCC 23365 (1 x 10 ⁷ CFU, IP).

Seven days after inoculation, the animals were divided into three groups with five mice in each of the 30 groups. Group 1 - untreated, control; Group 2 - (7 and 10 days post-inoculation) treated with MLV-encapsulated streptomycin sulfate (10 mg / kg body weight).

I.P.); MLV was prepared by standard techniques using 100 mg egg phosphatidylcholine (EPC) and 2 ml sterile HEPES in streptomycin sulfate (100 mg / ml). Lipid: Streptomy Cin Sulfate Ratio: 100 mg EPC: 28 mg Streptomy Cin Sulfate in 2 ml final volume MLV suspension; Group 3 - (7 and 10 days post-inoculation) treated with SPLV-enclosed streptomycin sulfate (10 mg / kg body weight IP), SPLV-enclosed streptomycin sulfate was prepared as described herein by the following modifications. : 100 mg EPC and 0.3 ml HEPES containing 100 mg streptomycin sulfate were used in the preparation. The lipid streptomycin sulfate ratio in SPLV was 100 mg EPC-28 mg streptomycin sulfate in a 2 ml final volume suspension.

Fourteen days after inoculation with B. canis, all animals were sacrificed, the spleen was aseptically removed, homogenized, and a series of dilutions was prepared and applied to brucella agar for isolation of B. canis. The surviving B. canis results for each organ (after four days incubation) are shown in Table IX. Table.

Effectiveness of treatment with various SPLV-encapsulated antibiotics in infections.

fifty adult male Swiss mice were inoculated with B. cani ATCC 23365 (1 x 10 ⁷ CFU, IP).

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191,858

Seven days after inoculation, the animals were divided into ten groups with five mice in each group. Group 1 - untreated, control; Group 2 - 7 and 10 days post-inoculation after treatment with buffer-saturated SPLV (0.2 ml I.P.); Groups 3, 4, 5 and 6 received 0.2 ml intraperitoneally of an aqueous injection of dihydrostreptomycin, gentamycin, kanamycin or streptomycin at 10 mg / kg body weight 7 and 10 days after inoculation (each of these antibiotics was Destroys B. canis). Groups 7, 8, 9 and 10 received 10 mg / kg body weight of dihydro-streptomycin, gentamycin, kanamycin, or streptomycin in SPLV 7 and 10 days after inoculation with B. canis all animals were sacrificed, the spleen was aseptically removed, homogenized, serially diluted, and subjected to brucella agar for isolation of B. canis. The results obtained are shown in Table X.

Table X.

Comparison of Inhibition of Various Antibiotics in B. canis in Viviv ³

Battery formation unit B, canis / spindle * ⁵ enclosed in SPLV

aqueous solution antibiotic untreated antibiotic ^c 3.93 + 1.51xl0 ⁶ 4.66 ± 0.87x10® Dihydro-Streptomyces neomycin 1.13 ± 0.30xl0 ⁵ 0 gentamicin 7.06 + 2.53xl0 ⁵ 0 kanamycin 2.72 ± 0.91xl0 ⁵ 0 streptomycin 1.01 ± 0.17x105 0

a) Intraperitoneal treatment, 10 mg / kg body weight, no control group

(b) The number of CFU isolates per spleen of surviving B. canis was determined for each organ.

c) Antibiotics inhibiting B. canis in suspension culture.

The results of treatment of mice infected with B. canis with various antibiotics are summarized in Table X. The results obtained show that antibiotics which showed inhibition of B. canis under in vitro conditions (i.e., in suspension culture) only showed inhibition of B. canis when encapsulated in SPLV. Treatment with either aqueous antibiotics or buffered SPLV, or no treatment at all, showed that no surviving B. canis could be isolated from the spleen tissue.

Treatment of dogs infected with B. canis

Adult female straight-footed hunts were challenged orally and vaginally with B. canis ATCC 23365 (1x10 ⁷ CFU). Seven days after inoculation, the dogs were divided into three groups. Group 1 - no treatment, control; Group 2 - (7 and 10 days ⁵ postinoculation) received aqueous streptomycin sulfate at a dose of 10 mg / kg body weight (dosing in 5.0 mL IP); Group 3 (7 and 10 days post-inoculation) received 10 mg fkg body weight strepptomy cin sulph ( ¹⁰ doses in 3.0 ml IP). At specific intervals, vaginal swabs and heparinized blood samples from dogs were collected. To isolate B. canis, swabs and blood samples were plated on brucella agar. The results obtained are shown in Table XI. Table. Before, during and at the end of the experiment, serum samples were collected for the detection of B. canis-induced serum antibody. These results are also reported in Table XI. Table. Twenty-one days after inoculation with B. canis, all animals were euthanized. The following tissues were aseptically removed, homogenized, and serially diluted to isolate B. canis on brucella agar: heparinized blood, vaginal secretion, lung, spleen, synovial fluid, uterus, ovary, knee lymph node, lymphatic gland, intestinal lymph nodes, bone marrow, superficial cervical lymph nodes ³⁰ nodes, complementary lymph nodes. The results obtained are shown in Table XII. Table summarizes.

Table XI

Results of culture and serological tests in dogs with B. canis in two-step antibiotic treatment ³

Number of days after closed infection with B. canis SPLV Control Streptomycin * ⁵ Streptomycin ^c

45 Eló'ke- R M B V R M B V R M B V Our Story 0 0 0 0 0 0 0 0 0 0 0 0 0 2 ND ND + + ND ND + 0 ND ND + + 4 ND ND + + ND ND + + ND ND + + 50 Utóke- Our Story 8 0 0 0 + 0 0 + 0 0 0 0 0 10 0 0 0 + 0 0 0 + 0 0 0 0 21 1.5 2 + 1 2 + 4 0 0 0 0

a) R (rapid ski agglutination test) for serum value B. canis antigen reciprocal (x10 ² ); 0 = undetectable value.

M (2-mercaptoethanol ethanol needle agglutination test) is the reciprocal of serum value of B. canis antigen (x10 ² ); 0 = undetectable value.

B (blood culture) and V (vaginal culture) on brucella agar: + = detection greater than or equal to 1 CFU; 0 = battery not detectable; the controls received no treatment.

-151 1I

b) Streptomycin sulfate (aqueous) 10 mg / kg body weight, I.P.

c) Streptomy cin sulfate 10 mg / kg body weight, enclosed in SPLV, I.P.

XII. spreadsheet

Tissue culture results of dogs infected with B. canis by two-step antibiotic treatment ³

Tissue * ⁵ Streptomycin enclosed in SPLV ^c Streptomycin ^d control blood 0 + + vaginal neck 0 + + lungs 0 + + spleen 0 + + synovial sap N. D. 0 0 bee 0 + + ovary 0 + + popliteal lymph node . N. D. + + salivary glands 0 0 0 almond 0 + + lymph nodes 0 N. D. + intestinal ripple lymph node N.D, 0 0 medulla 0 + + surface neck lymph node N. D. N. D. + additional' lymph nodes 0 + +

(a) Animals treated on days 7 and 10 of post-infection.

(b) serial dilutions of necropsycus samples from brucella agar; + = Greater than or equal to 1 CFU; 0 = no battery detected.

c) Streptomycin sulphate (10 mg / kg body weight, I.P.) enclosed in SPLV

d) Streptomycin sulfate (aqueous) (10 mg / kg body weight, I.P.).

e) Untreated controls.

The XI. Table 2 summarizes the results of culture and serological tests of dogs infected with B. canis in a two-step antibiotic treatment, representing values obtained before, during and after treatment. All experimental animals were serologically negative prior to exposure to B. canis, as indicated by negative serum values. Negative cultures were also obtained from blood cultures and vaginal mucus cultures. All experimental animals showed positive cultures in both blood and vaginal mucus cultures 7 and 10 days prior to treatment. Vaginal cultures of dogs treated with aqueous streptomycin or untreated dogs remained positive at day 21 at the end of the post-treatment period.

[858 2

Group 3 - Streptomy received cin-containing liposomes, the cultures were negative one day after the first treatment and remained negative during the post-treatment period. Dogs that were untreated or treated with aqueous streptomycin produced detectable serum levels against B. canis antigen on day 21 post-treatment. Experimental animals receiving 10 treatments of SPLV-enclosed antibiotics showed no detectable antibody production against B. canis antigen 7 and 10 days after inoculation. Test results of two-step antibiotic treatment of dogs infected with B. canis, shown in Table XII. Table 1A shows that only treatment with streptomycin enclosed in SPLV was so effective that B. canis was eliminated from tissue samples from all organs.

Treatment of guinea pigs infected with B. abortus Adult female guinea pigs were infected with a culture of B. abortus 25 ATCC 23451 (1 x 10 ⁷ CFU, IP).

Seven days after inoculation, the animals were divided into three groups so that each group had five animals. Group 1 - no treatment, control. Group 2 - received aqueous streptomycin sulfate treatment with 0.2 ml of 10 mg / kg body weight I.P. injection on the seventh and tenth day after inoculation with B. abortus.

Group 3 received treatment with Streptomycin Sulphate encapsulated in SPLV in a volume of 0.2 ml, 10 mg / kg I.P. 14 days after inoculation with B. abortus, all animals were sacrificed, the spleen was aseptically removed, homogenized, serially diluted, and subjected to brucella agar for isolation of B. abortus. Figure 7 shows the amount of B. abortus remaining in the spleen after four days of incubation. Only treatment with streptomycin enclosed in SPLV was effective in completely eliminating the remaining B. abortus in guinea pig spleen. Experimental animals that received or were not treated with aqueous streptomycin remained B. abortus fer50.

Treatment of cows infected with B. abortus

Nine severely infected animals were tested during the experiment B. abortus bacterial isolates were taken from milk, vaginal mucus and showed negative results after treatment with streptomycin for six weeks in SPLV. When the animals were re-infected, we could only isolate bacteria from those parts of the udder that were positive before treatment.

Nine crossed (hereford-jersey-Brangus) 22-month-old non-pregnant B. abortus positive cows were used in the experiments. At least 4 months before the commencement of the studies

-16XIII. spreadsheet

Results of culture of tissue samples from cows infected with B. abortus

191,858 experimental animals were challenged per conjunctiva with 1 x 10 ⁷ CFU of B. abortus strain 2308 in the middle of pregnancy, resulting in abortion and / or B. abortus positive lymphoid or uterine section and / or fetal tissue results. The cows were divided into three groups. The treatment was performed in two phases, the three groups being separated as follows: 1, - 3 cows received saline by intraperitoneal injection; 2, - three cows received intraperitoneal injection of aqueous antibiotics and buffered SPLV treatment (streptomycin was used as an antibiotic at a dose of 10 mg / kg body weight); 3, - 3 cows received SPLV-enclosed streptomycin treatment by intraperitoneal injection at a dose of 10 mg / kg body weight. The total injection volume per animal was 100 ml.

During the first two months, milk and bee secretion cultures were prepared. All experimental animals were euthanized by an overdose of sodium pentabarbitol and the following organs were harvested in duplicate bacterial culture. 1., lymph nodes, right and left atlantic, right and left suprafaryngeal, right and left mandibular, right and left perotid, right and left prescapular, right and left prefemoral, right and left axillary, right and left popliteal, right and left iliaÚs , right and left supramammlis, right and left renal, bronchial, mediastinal, mesenteric, and hepatic; 2., glands: all four quarters of the mammary gland, left and right mammary glands and thymus, if present; 3., organs and other tissues: spleen, liver, left and right horns of the uterus, cervix, vagina, kidney and tonsils.

After necroscopic examination, all tissues were frozen and stored at -70 ° C. The tissues were thawed, disinfected with alcohol and aseptically trimmed prior to measurement.

The weight of a tissue unit was 0.2 to 1.0 g. The tissue pieces were homogenized in 1 ml of sterile physiological saline, serially diluted with sterile physiological saline, and a homogenous suspension of 1:10 to ¹⁰ was prepared. An aliquot of 20 ml of each dilution series was taken and transferred to brucella agar and incubated at 37 ° C. Each tissue was examined twice.

The plates were examined daily and inoculated with bacterial culture. Colonies were isolated on day 3, inoculated, and identified by Gram staining. On days 5, 6, and 7 of incubation, colonies were counted and CFU / gram tissue was determined. The colonies were inoculated again for bacteriological confirmation of the assay.

Bacteriological isolates were taken from each tissue sample and the bacterial / gram tissue value was quantified. The results for the four animals tested - 1 placebo control and 3 animals treated with SPLV-incorporated streptomycin - are summarized in Table 13.

Fabric

Untreated control

Streptomycin incorporated into SPLV

Adrenal gland left Adrenal gland right Atlantic LN right Atlantic LN left Supplemental LN left Supplemental LN right Bronchialis LN Cervical Hepatic LN Uterine horn left Uterine horn right Inner hip LN right Inner hip left Kidney

Liver

Lungs

Mammary Gland LF Mammary Gland LR Mammary Gland RF Mammary Gland RR Laryngeal LN Lumbar Glandular LN Laryngeal LN Laryngeal LN Laryngeal LN

Supramammalian LN left Supiamammalian LN right Suprafarange LN left Suprafarange LN right Timus

Vagina ++ ++

-H-H ++ +

++

++++ +

ΊΟ

ΊΟ ο

ο ο

ο ο

ο ο

ο ο

ο ο

ο ο

ο ο

ο

ΊΟ ο

ο ο

ο ο

ο ο

ο ο

ΊΟ ο

ο ο

ο ο

-ΙΟ ο

ο

ΊΟ ο

ο ο

ο

ΊΟ ο

ο ο

ο ο

ο ο

ο ο

ο ο

ο ο

ο ο

ο +

+

-ΙΟ ο

ο ο

ο +

ο ο

ο ο

ο ο

ο

ΊΟ ο

ο ο

ο ο

ο ο

ο ο

ο ο

+ ο

ο ο

ο ο

ο ο

ο ο

LN ο

+

4 + Η

- lymphatic gland

In 0.3-1 g tissue culture, bacteria were undetectable to less than 200 colonies tissue to more than 300 colonies / g to more than 1000 colonies to more than 100,000 colonies / g

Treatment of eye injuries

Bacterial and similar eye infections and other eye injuries cause economic and public health problems worldwide. Untreated or inadequately treated eye diseases for loss of vision or blood poisoning17

-171 19 may lead to death. Infections of the eye in humans and animals include, but are not limited to, Clostridium spp., Corynebacterium spp., Leptospira spp., Moraxella spp., Mycobacterium spp., Neisseria spp., Propionibacterium spp., Proteus spp., Pseudomonas spp. Serretia spps., E. Coli spp., Staphylococcus spp., Streptococcus spp. and bacterial-like microorganisms such as Mycoplasma spp. and Rickettsia spp. Animals and humans can be bacilli, meaning that infections can be transmitted to each other.

Antibiotic treatment of the above bacterial infections is lengthy and inconvenient due to the frequency of treatment, sometimes every 20 minutes, and the unwanted high antibiotic concentration in the tissues. Longer treatment is difficult for a number of reasons. In many cases, infectious microorganisms on the surface of the eye tissue are highly resistant to antibiotic activity. Topical administration of antibiotics results in rapid drug withdrawal from the eye and results in variable contact times. As a general rule, eye infections should be fully treated, as residual infections can cause re-infection during tears and the infection cycle will resume. In many cases, therapeutic drug concentrations - the amount of drug needed to eliminate infections - lead to decreased vision and in some cases, complete blindness. Such pet diseases cause millions of dollars in losses every year. The only potential way to control such infections is through the advanced therapy and quarantine of the infections.

In the following experiments, the efficacy of treatment with free glycerol dissolved in glycerol in an eye infected with M. bovis and an antibiotic encapsulated in SPLV is compared and evaluated.

M. bovis causes keratoconjunctivitis in cattle (pink eye). The disease is characterized by blepharospasm, lacrimation, conjunctivitis, various degrees of corneal vision and ulceration. In adult cattle, there is a slight decrease in appetite and a decrease in milk yield. Although many antibiotics are effective in treating M. bovis, treatment should be given early and repeatedly by topical or subconjunctival injection.

As described above, the efficacy, duration of action, and mode of action of the therapeutic agent are provided. Surprisingly, it has been found that treatment once or twice with the therapeutic systems of the present invention is effective for such infections as opposed to insufficient treatment with conventional antibiotics. Conventional antibiotic therapy is inadequate because there are infections remaining in the eye that re-infect the eye, a new cycle of infection occurs, and the infection can be completely overcome only by repeating the antibiotic therapy several times.

ί 858 2

Treatment of mouse keratoconjunctivitis infection

160 C 57 black mice were divided into eight groups. Half of each group received UV radiation in both eyes to develop corneal insufficiency. The right eye of each animal was then infected with M. bovis with a bacterial concentration of 1 x 10 ⁶ le10 in the eye. After 24 hours of incubation, all animals were tested for corneal opacity. Eight groups were treated locally on each eye as follows: Groups 1 and 2 received 10 μΐ streptomycin (30 mg / ml) in SPLV, Groups 3 and 4 received 10 μΐ streptomycin (100 mg / ml); Groups 5 and 6 received 10 pl of buffered saturated SPLV (100 mg / ml) in aqueous streptomycin; groups 7 and 8 received 10 μΐ sterile saline (untreated left eyes treated with the same topical solutions to investigate the potential eye irritation of SPLV; no irritation was observed). Once a day, the animals were examined for progression or regression of comical vision.

On the 3rd, 5th and 7th days of post-treatment, the right eye was swabed and the isolates obtained were obtained. and bovis. M. bovis colonies were characterized morphologically and by fluorescence-labeled antibody reactivity. The results obtained a

XIV. Tables 1 to 4 show that, when evaluated, we found that only streptomycin incorporated into SPLV was capable of controlling the infection.

Treatment of rabbit conjunctiva with anti35 biotics enclosed in SPLV

M. bovis strain ATCC 10900 was diluted in sterile physiological saline (0.085% NaCl) to a concentration of 1 x 10 ⁷ cells / ml. A 0.1 ml aliquot of the bacterial suspension was used to infect locally the eyes of 10 adult female rabbits. The cultures were sampled daily by swabing and the conjunctival material was placed on a blood agar le45 to isolate M. bovis. Three days after infection, the rabbits were divided into three groups: two animals (control) received no treatment; four animals received sterile saline streptomycin (concentration: 10 mg / kg body weight); four animals received streptomycin incorporated in SPLV (concentration: 10 mg streptomycin / kg body weight) in ste50 ril physiological saline. All solutions were administered topically to both eyes. After 24 hours, the tampon of each rabbit conjunctiva was repeated and performed daily for seven days. The results obtained with M. bovis isolates on blood agar plates are shown in Table XV. Table.

-182

Results of treatment of keratoconjunctivitis in mice infected with M. bovis

191,858

XIV. spreadsheet

Mouse number / Group of 20 mice M. bovis culture ³ Pretreatment Batting after treatment days Cornea Cornea number blur ¹⁵ blur ¹¹

No irradiated mice 0 1 2 3 4 0 1 2 3 4 3 5 control 16 3 0 1 0 18 2 0 0 0 4/5 4/5 Free streptomycin ^c 18 1 1 0 0 18 2 0 0 0 2/5 2/5 Buffer-saturated SPLV plus free streptomycin® 17 2 1 0 0 18 1 1 0 0 2/5 3/5 Streptomycin® enclosed in SPLV 17 3 0 0 0 20 0 0 0 0 0/5 0/5 UV irradiated mice control 1 1 5 9 4 10 3 1 2 4 5/5 5/5 Free Streptomycin ⁰ 0 4 9 7 0 14 3 2 1 0 3/5 4/5 Buffer-saturated SPLV plus free streptomycin ⁰ 0 3 5 10 2 11 2 4 3 0 3/5 3/5 Streptomycin® enclosed in SPLV 0 1 5 11 3 19 1 0 0 0 0/5 0/5

Notes to Section XIV. Table ^of M. bovis positive aspects of cultures. The determination is carried out by the fluorescent antibody spot method.

^b Normal cornea: 1 - less than normal light 2 = slight focal haze; 3 = partial opacity of the cornea; 4 = total corneal opacity.

^c 10 µl total dose (1.0 mg streptomycin / eye).

XV. spreadsheet

Test results of rabbit conjunctival specimens after local M. bovis infection followed by treatment with SPLV and water-soluble streptomycin

M. bovis cultures ³ Animal Post infection days Group song Pre - treatment ¹³ Batting 1 2 3 4 5 6 7 control 1 0 + +++ + + 2 0 + +++ + + streptomycin ^ 1 0 4- +++ + + 2 0 0 +++ + + 3 0 + +++ + + 4 0 + +++ + + Locked in SPLV 1 0 0 + 00 0 0 streptomycin ^e 2 0 + + 00 0 0 3 0 + + 00 0 0 4 0 + + 00 0 0

Notes to Section XV. table:

^a Cultures are established by plating M. bovis colonies on blood agar plates and incubating at 37 ° C for 24 hours. Plus (+) indicates value = 1 CFU for M. bovis / isolate ratio; 0 = no colonies were detected.

^b All animals were infected locally with 1 x 10 ⁶ CFU M. bovine cultures in both eyes.

^c Animals were treated locally with 0.1 ml solution in both eyes.

^d Streptomycin (10 mg / kg body weight) in sterile physiological saline.

^e Streptomycin (10 mg / kg body weight) enclosed in SPLV in sterile physiological saline.

-191

191,858

Treatment of keratoconjunctivitis caused by subcutaneous infections

M. bovis, ATCC 10900 was diluted to 1 x 10 ⁷ cells / ml in sterile physiological saline. An aliquot of 0.1 ml of the bacterial suspension was used to infect adult rabbit eyes, which had been previously infected with SPLV as described above. The right eye of each rabbit was subcutaneously infected with 0.1 ml of M. bovis in connective tissue, and the left eye of each animal was locally infected with 0.1 ml of M. bovis. Cultures of connective tissue from each eye of each animal were taken daily and plated on blood agar plates to isolate M. bovis. 3 days after inoculation, rabbits were divided into three groups: two animals were untreated; three animals received streptomycin in a standard ophthalmic glycerol suspension (streptomycin concentration: 10 mg / kg body weight); four animals received a suspension of streptomycin (10 mg streptomycin sulfate) per kg body weight in a physiological suspension. The suspension or solution was applied topically to each eye in a volume of 0.1 ml. After 24 hours and on each of the following five days, the rabbit conjunctiva was sampled. The results obtained with M. bovis isolates obtained on blood agar plates are shown in Table 16. At the end of the experiments, all animals were dissected and the conjunctiva was removed. Samples were vascularized, dissected, homogenized and plated on blood agar plates to isolate M. bovis. The results obtained are shown in Table XVII. Table.

Comparison of efficacy of SPLV and liposome formulations in the treatment of ocular infections

M. bovis (ATCC 10900 strain was diluted in saline to 1 x 10 ⁷ cells / ml in saline solution). A 0.1 ml aliquot of bacterial suspension was used to subcutaneously infect the connective tissues of both eyes of adult rabbits. 5 days post-infection, the rabbits were divided into 6 groups: 2 animals received no treatment (control), 3 animals received SPLP-encapsulated streptomycin suspension (10 mg streptomycin sulfate / kg). body weight), the suspension was diluted 100-fold, OD _48C =

0.928 (optical density at 480 mm) was set; three animals received SPLV encapsulated streptomycin suspension (10 mg streptomycin sulfate / kg body weight) diluted 100-fold and adjusted to OD ₄₈₀ = 0.449; three animals received a suspension of streptomycin (10 mg streptomycin sulfate / kg body weight) in SPLV, diluted 100-fold with OD ₄₈₀ = 0.242; three animals received a suspension of streptomycin (10 mg streptomycin sulfate / kg body weight) encapsulated in SPLV, diluted 100-fold and adjusted to an OD of _{48 (} , = 0.119; two animals received multilayer 5 secular streptomycin (MLV)). Streptomy cin sulfate (10 mg / kg body weight) was diluted 100-fold and adjusted to OD480 -0.940r MLVs were prepared according to the procedure described by Fountain et al., Curr. Micro 6: 373 (1981). , by adding streptomycin sulfate to the dried lipid film, which was then stirred and swelled for two days, and the unincorporated streptomycin was removed by repeated centrifugation.

XVI. spreadsheet

Test results of M. bovis post-infectious isolates on conjunctival membrane treatment with ophthalmic glycerol streptomycin solution or saline containing streptomycin encapsulated in SPLV

Group Animal szám6 M. bovis cultures ³ Post infection days Pretreatment Batting 1 2 3 4 5 control 1 + + + + + 2 + + + + + Glycerine strepto- 1 4- + + + + gums solution ^ 2 + + + + + Get into SPLV 3 + + + + lízott 1 + 0 0 0 streptomycin ^e 2 -1- + 0 0 0 3 + + 0 0 0 4 + 0 0 0

Notes to Section XVI. table:

^a Cultures are established by plating M. bovis colonies on blood agar plates and incubating at 37 ° C for 24 hours. (+) indicates that value = 1 CFU M.

⁵⁰ bovis / isolate ratio; 0 = no colonies were detected.

^b All animals were infected locally with 1 x 10 * CFU of M. bovis in both eyes; in the connective tissue membrane of the right eye 1 χ 10 ⁶ CFU

M. bovis was injected; locally to the left eye 1 x 10® CFUM. bovis was added.

^c Animals were treated locally with 0.1 ml solution in both eyes.

^d The animals were treated locally with a solution of glycerol streptomycin (10 mg / kg body weight) locally in each eye.

^e Animals were treated with sterile physiological saline (10 mg / kg body weight ⁶⁵ ) locally encapsulated in SPLV in both eyes.

-201

XVII. spreadsheet

Dissection results for ocular and related tissue in rabbits after conjunctival infection with M. bovis and treatment with ocular glycerol streptomycin solution or sterile saline containing streptomycin encapsulated in SPLV

M. bovis cultures Isolation Vascularization of the right eye Control A + 2+ B + 2+ Glycerin streptomycin solution A + 2+ B + 1+ C + 2+ D + 2+ Streptomycin encapsulated in SPLV THE 0 0 B 0 0 C 0 0 D 0 0

191,858

The suspension was applied topically to both eyes. After 24 hours, all rabbits were excreted for conjunctival secretion (for 9 days daily) and placed on blood agar. The results of the isolation of M. bovis ^{5 on} blood agar plates are shown in Figure XVIII. Table. All animals were autopsied. Lacrimahs secretions and conjunctivae were removed aseptically from all animals. These organs were vascularized with ¹⁰ needles, minced, homogenized, and plated on blood agar plates to isolate M. bovis. The results are shown in Table 19.

^the explanation is in Annex XII. Table 5, performed on day 5 post infection.

^b Vascularization as follows: 0 = normal vessels, 1 = some vessels markedly dilated and infiltrated compared to small vessels; 2 = red diffusion not readily detectable by individual vessels; 3 = strong red diffusion, vascular fission, and blood effusion in the conjunctiva.

XVIII. spreadsheet

Isolation of M. bovis after treatment with physiological saline containing streptomycin diluted in SPLV diluted in conjunctiva of infected rabbits or physiological saline containing streptomycin encapsulated in MLV

Isolation of M. bovis ³ Post-infection days

Group Animal Pretreatment Aftertreatment

song 1 2 3 4 5 6 7 8 9 10 11 12 13 14 control 1 + 4 + + + + 4 + + + + + + + 2 + + + + + + + + + + + + + + Encapsulated in MLV 1 + 4- -1- + + + 1- 0 0 + + + 0 + streptomycin 2 + + + + + + 0 + + 0 0 + + 0 It is encapsulated in SPLV 1 + 4 ^- + + -1- 0 0 0 0 0 0 0 0 0 streptomycin 2 + + + + + 0 0 0 0 0 0 0 0 0 (Undiluted) 3 + + + + + 0 0 0 0 0 0 0 0 0 It is encapsulated in SPLV 1 + + + + + + 0 + + + -1- + + + streptomycin 2 + + + + + 0 0 + 0 0 0 0 0 0 (1: 2 dilution) 3 + + + + 0 0 0 0 + 0 0 0 0 It is encapsulated in SPLV 1 + + + + + + + 0 0 0 0 0 0 0 streptomycin 2 + 4- + + + + 0 0 0 + 0 0 0 0 (1: 4 dilution) 3 + + . + + + 0 0 + + + + + + + It is encapsulated in SPLV 1 -1- + + + + 0 0 + + + + + + + streptomycin 2 + + + + + 0 0 0 0 0 0 0 0 0 (1: 6 dilution) 3 + + + + + + + + 0 + + + + +

All ^the animals were infected with 1 x 10 ⁶ CFU M. bovis such a way that it is injected into each eye conjunctiva membrane. The conjunctival mucosa was placed on blood agar. Cultures were established by placing M. bovis colonies on blood agar lysis and incubating for 24 hours at 37 ° C. (+) = = 1 CFU; 0 = no cultures were detected.

-21191 858

XIX. spreadsheet

Necropsy results of rabbit eyelid and surrounding tissue after treatment with M. bovis conjunctival infection and treatment with dilutions of MLV-encapsulated streptomycin, SPLV-encapsulated streptomycin, or SPLV-encapsulated streptomycin.

Animal Isolation of M. bovis The eyes vaszkula- rizációja Lacrimal deposition ^c control 1 + 1+ 1+ 2 + 1+ 1+ Encapsulated in MLV 1 + 1+ 1+ streptomycin 2 0 1+ 0 It is encapsulated in SPLV 1 0 0 0 streptomycin 2 0 1+ 0 (Undiluted) 3 0 0 0 It is encapsulated in SPLV 1 + 2+ 2+ streptomycin 2 0 0 0 (1: 2 dilution) 3 0 1+ 0 It is encapsulated in SPLV 1 0 0 0 streptomycin 2 0 1+ 0 (1: 4 dilution) 3 + 1+ 0 It is encapsulated in SPLV 1 + 1+ 1+ streptomycin 2 0 1+ 0 (1: 6 dilution) 3 + 1+ 0

Notes to XIX. For Table 1, ^the Explanation is the same as for Table XIV, post infection on Day 14.

^b Vascularization as follows: 0 = normal blood vessels; 1 = some dilated vessels infiltrated with small blood vessels; 2 = diffusely red with hardly detectable individual vessels; 3 = Strong red diffusion, vascular fission, and blood effusion into the conjunctiva.

^C Deposition as follows: 0 = no deposit, 1 = eyelid and eyelash dirt deposit; 2 = deposits of eyelid and eyelashes around the eyes.

Treatment of viral infections

Lymphocyte choriomeningitis virus (LCMV) is a member of the Arenavirus family, causing human disease and fatal LCMV infection in mice by intracerebral infection. The cause of death in mice is that the immune cells react with the virus-infected cells. The virus does not kill the cell in which it multiplies, and therefore the therapeutic eyes used in the mice should inhibit viral growth in such a way that immune cells are not activated and / or inhibit the activation of immune cells. The efficacy of treating viral infections with antiviral compounds encapsulated in SPLV is shown below.

Treatment of lethal lymphocyte choriomeningitis virus infections in mice Monthly Swiss mice were infected intracerebrally with a lethal dose of LCM virus (100 colony forming units (PFU) per 0.5 ml inoculum). The mice were divided into four groups, each containing two mice, each of which received 2, 3 and

On day 4, it was treated by intraperitoneal injection (0.1 ml / dose / mouse) as follows: 1. * The "SPLV group" was treated with 3 mg Ribavarin / ml suspension in egg phosphatidylcholine SPLV. SPLV was prepared using 100 mg of lipid and 100 mg of active ingredient / ml in 0.3 ml PBS buffer. The incorporation rate was 10%. 2. Group R was treated with Ribavarin in PBS 3 mg / ml; 3. The SPLV group was treated with buffer saturated SPLV (SPLV was prepared as above except that Ribavarin was not used); 4. The "control group" was treated with PBS. On day 5 post-infection, two mice from each group were sacrificed and their spleen homogenized (2 spleen / group homogenized in PBS using 1/20 w / v buffer). Platelet forming units (PFU) were determined per ml in each suspension. The remaining five mice were observed twice daily for 30 days until death of the entire group.

The results obtained are shown in Table XX. Table.

In the XX. Table 1 clearly illustrates the reduction in lethality and the decrease in the number of viruses recovered from infected animals. However, it has not been determined whether these results are due to the antiviral activity of Ribavarin lysed from SPLV or to immunomodulation of the virus host mouse (Ribavarin liberated from SPLV).

XX. spreadsheet

Treatment of Lethal LCM Virus Infection in Mice ⁴

Group Stroke ^b Virus recovered from L intact (PFU x 10 ³ / ml) ^c control 5/5 7.0 SPLV-group 5/5 6.9 Group R 5/5 5.2 SPLV-R group 5/5 3.4

^two- month-old mice were each challenged intracerebrally with a lethal dose (100 PFU LCM virus in 0.05 ml inoculum).

^b Fatalities are expressed as the number of deaths / dwindling number.

^c On day 5 post-infection, two mice from each group were sacrificed, the spleen was removed, homogenized and a homogenate of 1 g spleen / 20 ml was prepared.

Claims (15)

Claims CLAIMS 1. A method for producing stable plurilamellar vesicles of 100 to 10,000 nm, substantially free of multilamellar, unilamellar, and reverse-phase evaporative vesicles, wherein the vesicles comprise a few hundred ordered lipid bilayers of supramolecular structure containing aqueous soluble solids. around, characterized by 1. dispersing at least one amphipathic lipid, preferably a phospholipid, in an amount of 20 to 3000 mg / ml in an organic solvent, optionally containing an antioxidant, at a reaction temperature below the boiling point of the organic solvent, 11. mixing the resulting dispersion with an aqueous phase containing from 20 to 50% by weight of the active ingredient or other compound in a ratio of 3: 1 to 40: 1, and 111, the aqueous phase is emulsified and the organic solvent is simultaneously evaporated from the biphasic system. 2. The process of claim 1 wherein the lipid is phosphatidylcholine. 3. A process according to claim 1 wherein the antioxidant is an organic solvent. 4. A process according to claim 1 wherein the antioxidant is butylhydroxytoluene. 5. The process of claim 1 wherein the aqueous phase is a solute. 6. The method of claim 1 wherein the aqueous phase comprises an anti-bacterial, antifungal, anti-parasitic, or antiviral agent. 7. The method of claim 1, wherein the aqueous phase is an antitumor agent, a toxin, a cell receptor binding molecule, or an immunoglobulin. 8. The method of claim 1, wherein the active ingredient in the aqueous phase is an anti-inflammatory compound, a glaucoma compound, a pupil dilator, or a local anesthetic compound. ~ 9. The method of claim 1, wherein the aqueous phase is selected from the group consisting of an enzyme, a hormone, a neurotransmitter, an immunomodulator, a nucleotide, or a cyclic adenosine monophosphate. 10. The process of claim 1, wherein the aqueous phase is a solute Dye, fluorescent compound, radioactive compound or radiolabel compound are used. A process according to claim 1, wherein the process is carried out at a temperature of from 4 ° C to 60 ° C. 12. The process of claim 1, wherein said step is performed at a phase transition temperature of at least one lipid, 13. The process of claim 1, wherein the organic solvent is a fluorinated hydrocarbon or diethyl ether, or a mixture thereof. 14. The method of claim 1 wherein the lipid is a phospholipid. 15. The process of claim 14, wherein the pathway is a phospholipid with a twin ionic structure.